Microbes are known to have developed the ability to evolve different mechanisms of self-defense against antimicrobial agents. In particular, bacterial and fungal pathogens have developed mechanisms of resistance to antibiotics and antimicrobial agents used to inhibit their growth, or to treat infections by them in humans, animals and tissue cultures. As a result, treatment regimens can be adversely affected or, in some cases rendered ineffective.
One of the most frequently employed resistance strategies in both prokaryotes and eukaryotes is the transmembrane-protein-catalyst extrusion of drugs from the cell, with these proteins acting like bilge pumps, reducing the intracellular drug concentration to subtoxic levels. (M. Ines Vorges-Walmsley et.al., Trends in Microbiology, 2001, 9: 71–79).
Recent developments in the efflux area include the discovery of new monodrug efflux systems, as well as the realisation of the importance of multidrug efflux systems (H. Nikaido and H. I. Zgurskaya, Current Opinion in Infectious Diseases, 1999, 12: 529). In gram-negative bacteria, for example, single component efflux pumps extrude their substrates into the periplasmic space as is done by the transposon-encoded tetracycline- and chloramphenicol-specific pumps, TetA and CmlA, respectively, and the MDR pump MdfA encoded in the chromosome of Escherichia coli. 
Bacterial genomes sequenced to date almost invariably contain genes apparently coding for multidrug efflux pumps. Multidrug efflux as a major cause of intrinsic drug resistance in many microorganisms, or overproduction of intrinsic pumps, or acquisition of pump genes from external sources, all play a prominent role, often resulting in high levels of resistance. Examples of multicomponent efflux pumps, belonging mainly to the resistance-modulation-division (RND) family members, found mostly in gram-negative bacteria, include the MDR pumps AcrAB-TolC and MexAB-OprM from E. coli and Pseudomonas aeruginosa. Interplay between efflux pumps may provide either additive or multiplicative effects on drug resistance (A. Lee et al., J. of Bacteriology, 2000, 182: 3142). MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM, AcrAB-TolC, AcrEF, MarA, SoxS, or/and Tet pump/s are known to be present in Gram negative organisms such as P. aeruginosa and E. coli and are reviewed in recent publications and papers, such as K. K. Y. Wong et. al., J. of Bacteriology, 2001, 183: 367–374; K. Poole, Antimicrobial Agents and Chemothero, 2000, 44: 2233–2241; R Srikumar et. al., Abstracts of the 40th Interscience Conference on Antimicrobial Agents and Chemother., 2000, Abstr. 441: 74; Xian Zhi Li et. al., Journal of Antimicrob. Chemother., 2000, 45: 433–436; Koronakis et. al., Nature, 2000, 405: 914 –919; M Oethinger, et. al., Antimicrob. Agents and Chemother., 2000, 44: 10–13; W. V. Kern, et. al., Antimicrob. Agents and Chemother., 2000, 44: 814–820; O Lomovskaya, et. al., Antimicrob. Agents and Chemother., 1999, 43: 1340–1346. Active efflux has been shown to be the primary or molecular mechanism of a fluoroquinolone resistance in Salmonella enterica Serovar Typhimurium (E Giraud et. al., Antimicrob. Agents and Chemother., 2000, 44: 1223–1228) and in Pseudomonas aeruginosa isolates from cystic fibrosis patients (S Jalal et. al., Antimicrob. Agents and Chemother., 2000, 44: 710–712). Efflux mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei, the causative agent of meliodosis has been described in R A Moore et. al., Antimicrob. Agents and Chempther., 1999, 43: 465–470.
Efflux transporters are also among different mechanisms responsible for the resistance to antibiotics displayed by gram-positive bacteria and mycobacteria, particularly aerobic gram-positive cocci. The multidrug transporter NorA, belonging to the major facilitator superfamily (MFS) transporters, contributes to the resistance of Staphylococcus aureus to fluoroquinolone antibiotics. A minireview describes effllux-mediated resitance to fluoroquinolones in Gram-positive bacteria and the mycobacteria (K Poole, Antimicro. Agents and Chemother., 2000, 44: 2595–2599). To the MFS transporters also belong the Bmr and QacA/QacB efflux pumps of Gram positive bacteria, and EmrB of E. coli (H. Nikaido, Current Opinion in Microbiology, 1: 516–523). A structure based mechanism for drug binding by multidrug transporters is recently proposed using the BmrR protein from Bacillus subtilis and the multidrug transporter MdfA from E. coli (E. E. Zheleznova et. al., Trends in Biological Sciences 2000, 25: 39–43), which mechanism is also putatively considered for the QacA/QacB efflux system of S. aureus . New MFS pumps include PmrA (a homolog of NorA) of Streptococcus pneumoniae (J. Broskey etl.al., Journal of Antimcro. Chemother., 2000, 45: Suppl. S1, 95–99; M J Gill et. al., Antimicrob. Agents and Chemother., 1999, 43: 187–189) and Tap of Mycobacterium fortuitum and M. tuberculosis. Recently, NorM, which pumps out fluoroquinolones and some cationic agents was found to be outside the MFS transporter class. The role of membrane-fusion protein (MFP) structural homologues recently identified in low G+C Gram positive bacteria that lack an outer membrane, as essential transport accessory proteins with transporters of the ATP-Binding Cassette (ABC) super family has been described (K. T. Harley et. al., Molecular Microbiol., 2000, 38: 516–517).
The Mef efflux system mediates large fractions of erythromycin-resistant clinical isolates of S. pneumoniae (N. J. Johnston et al., Antimicrob Agents Chemother., 1998, 42: 2425; T. Nishijma et al., J. Antimicrob Chemother, 1999, 43: 637). Beta-haemolytic Streptococci and pneumococci resistant to erythromycin due to the prsence of MefA efflux pumps in Streptococcus pyogenes, MefE pumps in S. pneumoniae, and an M phenotype bearing S. agalactiae possessing MefA or MefE pumps are found to be emergent and prevalent in Europe (C Arpin et. al., J. Antimicrob. Chemother., 1999, 44: 133–138; E. Giovanetti et. al., Antimicrob. Agents and Chemother., 1999, 43: 1935–1940).
Efflux and drug resistance in fungi and protozoa have also been described (T. G. White et al., Clin Microbiol Rev 1998, 11: 382; D. Sanglard, Drug Resistance Updates1998, 1: 255; B. Papadopoulou et al., Drug Resistance Updates 1998, 1: 266; E. Orozao et al., Drug Resistance Updates, 1999, 2:188).
In summary, the above discussion indicates that cellular factors affecting transport (both active and passive transport) of antibiotics into bacterial cells are important components of antibiotic resistance for many microbial species.
One strategy to target resistance mechanisms of microbial self-defense is to find inhibitors of microbial efflux pumps and, in particular of bacterial and fungal efflux pumps.
There is disclosed in PCT Patent publication WO 96/33285 and U.S. Patent application U.S. Pat. No. 5,989,832 with priority in U.S. application Ser. No. 08/427,088 and PCT/US96/05469, methods for screening for inhibitors of microbial efflux pumps, efflux pump inhibitors, compositions containing such efflux pump inhibitors, and methods for treating microbial infections using those compositions, but unlike the efflux pump inhibitors of the present invention, the efflux pump inhibitors disclosed are dipeptide amides specifically inhibiting a Pseudomonas aeruginosa-type efflux pump. There is disclosed in PCT Patent publication WO/001714, with priority in U.S. application Ser. No. 09/108,906, compounds which have efflux pump inhibitor activity, methods of using such efflux pump inhibitors and pharmaceutical compositions which include such compounds, but unlike the efflux pump inhibitors of the present invention, the efflux pump inhibitors disclosed are dipeptide amide derivatives demonstrating pump inhibitory activity against P. aeruginosa strains containing singular efflux pumps and multiple efflux pumps.
Patent WO 99/17791, with U.S. priority in 60/060,898 discloses a method for inhibiting the selection or propagation of a bacterial mutant that overexpresses an efflux pump wherein the inhibitor disclosed is the dipeptide amide, L-phenylalanyl-L-arginyl-beta-naphthylamide (MC′-207,110), which is unlike the efflux pump inhibitor compounds of the present invention.
U.S. Pat. Nos. 6,245,746, 6,114,310 and WO 9937667 all with U.S. priority in U.S. application Ser. No. 09/012,363 disclosed methods of using efflux pump inhibitors which increase the susceptibility of microbes in particular P. aeruginosa strains to antimicrobial agents and pharmaceutical compositions which include such compounds.
U.S. Pat. No. 6,204,729 describes peptidomimetic efflux pump inhibitors; secondary amide containing benzoxazole derivatives, methods of using such efflux pump inhibitor compounds and pharmaceutical compositions which include such compounds.
Patent WO 00/32196, with U.S. priority in 60/110,841, discloses inhibitors of multidrug transport proteins which may be used in combination with existing antibacterial agents and/or antifungal agents, wherein the inhibitor is an indole or a urea or an aromatic amide or a quinoline, all of which inhibitors are unlike the efflux pump inhibitor compounds of the present invention. In addition, the inhibitors disclosed in patent WO 00/32196 are specifically inhibitors of bacteria expressing a norA pump, or a fungus expressing a multidrug transport protein.
Novel inhibitors of the NorA multidrug transporter of S. aureus having structurally diverse chemical structures were also described by P. N. Markham et al., (Antimicrob. Agents & Chemother., 1999, 43: 2404), among which the more active compounds include (a) those containing an indole moiety like the previously known inhibitor, reserpine, (b) biphenyl urea derivatives, (c) a substituted pyrimidinone derivative and (d) compounds INF 240 and INF 277, but they are all unlike the efflux pump inhibitor compounds of the present invention.
Another inhibitor of the NorA MDR pump in a pathogenic S. aureus strain is 5′-methoxyhydnocarpin (F. R. Stermitz et al., Proc. New York Acad. of Sci., 2000, 97: 1433), which has a structure unlike the efflux pump inhibitor compounds of the present invention.
Nocardamin, a cyclopeptide, was found to be a general antagonist of a tetracycline efflux pump from S. aureus . It has a structure unlike the efflux pump inhibitor compounds of the present invention.
Minocycline and 1,1-dimethyl-5-(1-hydroxypropyl)-4,6,7-trimethylindan (Ro 07-3149) inhibit the active tetracycline efflux pump in S. aureus 743 (T.Hirata et al., Biol Pharm Bull, 1998, 21:678). Both the compounds have a structure unlike the efflux pump inhibitor compounds of the present invention.
(±)-3-(2-chloro,5-bromophenyl) ethyl-4-fluoropiperidine as an example of 3-arylpiperidines has recently been described as potentiator of existing antibacterial agents against E. coli (A. Thorarensen et. al., Bioorg. and Medicinal Chem. Letters, 2001, 11: 1903–1096). The 3-arylpiperidine compounds have a structure unlike the efflux pump inhibitor compounds of the present invention.
To our knowledge no drug like organic molecule has been identified, described or proposed as an inhibitor of Mef efflux pump.
Deficiencies abound in the efflux pump inhibitors disclosed in the art prior to our present invention.
Reserpine is not a usable compound for therapy because of its neurotoxicity at the concentration required for efflux pump inhibition.
The inhibitors of single drug and multidrug transporters such as the dipeptide amide, MC′-207,110, are broad in specificity, inhibiting all three RND systems of P. aeruginosa involved in fluoroquinolone efflux, but have not been shown to be effective against pumps of other strains, for instance a NorA pump. Moreover, the methods employed to demonstrate their efflux pump inhibitory properties are mainly in vitro methods. For demonstration of in vivo activity, recourse has had to be taken to parenteral administration (cf. T. E. Renau et al, J. Med. Chem.,1999, 42, 4928), rather than oral administration, as it is generally known that oral bioavailability is poor for compounds of a peptidic nature.
The series of inhibitors described in WO 00/32196 and by Markham et al (vide infra) have been minimally and incompletely profiled in terms of their efficacy, safety and tolerability, which has yet to be demonstrated in in vivo models.
Potent inhibitors of microbial efflux pumps is thus an important goal for the improved control of infectious diseases, allowing a renaissance for drugs that are no longer effective owing to their efflux (K. Poole, Journal of Pharmacy and Pharmacology, 2001, 53: 283–294). The current inventors have synthesised screened and identified novel inhibitors of cellular efflux pumps of microbes. Distinctive structural features characterise the difference sets of efflux pump inhibitors for different microorganisms as will be described in the following description.